The majority of separations employing high-pressure liquid chromatography (HPLC) are performed in the so-called reversed-phase liquid chromatographic (RPLC) mode. In this mode, the column-packing material is referred to as the stationary phase. In RPLC the stationary phase is typically non-polar. The eluent, also referred to as the mobile phase, used to elute the various components from the stationary phase is relatively polar. It can include, for example, an aqueous buffer or a mixture of water and an organic solvent, e.g., an alcohol. Its polarity can be changed by increasing the concentration of the less polar liquid (the alcohol) in the mobile phase, a technique known in the art.
Reversed-phase HPLC is finding increased use in the area of bioprocessing because of HPLC's great ability to separate and purify biological materials. At the preparative scale, there are many unique considerations not applicable at the analytical scale. One such consideration is the need to sterilize a chromatography column prior to its use in the purification of a product intended for biological or human use. Another is the desirability of using larger particles, typically greater than 20 microns (i.e., micrometers or μm) in average particle diameter.
Among solid support materials, including synthetic organic polymers and metal oxides such as silica, alumina, titania, and zirconia, silica is by far the most widely used support for HPLC and almost the exclusively used support for RPLC stationary phases. The high mechanical stability, monodisperse particle sizes, high surface area, and easily tailored pore size distributions make silica an ideal choice for efficient analytical RPLC columns.
Silane bonding chemistry also allows for a wide variety of stationary phases with different selectivities to be made on silica. The most commonly used stationary phases feature a non-polar ligand covalently bound to a porous silica particle through one or more siloxane bonds (Si—O—Si) to render the surface hydrophobic. The most familiar type of silica-based RPLC stationary phase is the dimethyloctadecylsilane bonded phase.
Although these conventional silica-based bonded phases are very useful for a wide range of applications in RPLC, their routine use is limited to the pH range of between about 2 and 8. The poor stability of conventional columns under low pH conditions seriously inhibits the use of pH as a mobile phase variable in separation optimization. Low pH mobile phases hydrolyze the siloxane bond between the bonded silane and the silica surface resulting in a continuous loss of chromatographic retention and the attendant irreproducibility in performance.
Thus, it is particularly desirable to have an acid-stable silica-based material that can be used with acidic mobile phases. Low pH mobile phases are particularly useful for the HPLC separation of a wide variety of silanophilic solutes such as basic drugs, peptides, and proteins and they have found wide use when HPLC is combined with mass spectrometric detection. At sufficiently low pH, acidic mobile phases suppress or completely eliminate deleterious interactions between positively charged solutes and the surface by protonating the surface silanol groups. This often results in peak shapes and efficiencies far superior to that achieved for the separation of the same solutes with neutral mobile phases.
As mentioned above, in addition to the use of a pH-stable support material, the production of a stable, reversed-phase material also requires a process for modifying the support material, which results in a stable, hydrophobic surface. Silylation is the most widely used method to derivatize silica particles to produce hydrophobic reversed-phase supports.
A synthetic approach for enhancing the low pH stability of silica-based bonded phases has been achieved using a bulky silane to sterically protect the silane stationary phases as disclosed in J. J. Kirkland, Analytical Chem., 61, 2-11 (1989); U.S. Pat. No. 4,705,725 (Glajch et al.); and U.S. Pat. No. 4,847,159 (Glajch et al.). Such sterically protected material is schematically shown in FIG. 1A. A synthetic approach for enhancing the high pH stability of silica-based bonded phases has been achieved using a bidentate silane with extensive endcapping as disclosed in J. J. Kirkland, Analytical Chem., 61, 2-11 (1989); and U.S. Pat. No. 4,746,572 (Glajch et al.). Such material is schematically shown in FIG. 1B.
A synthetic approach for enhancing both the high and low pH stability of silica-based bonded phases has been achieved using a self-assembled monolayer (SAM) of alkyl chains with siloxane bonding between the self-assembled moieties in the SAM layer as well as siloxane bonds between the SAM layer and the silica as disclosed in R. W. P. Fairbank et al., Analytical Chem., 67, 3879-3885 (1995); U.S. Pat. No. 5,716,705 (Wirth et al.); and U.S. Pat. No. 5,599,625 (Wirth et al.). Such material is schematically shown in FIG. 1C.
An alternate approach to silylation for modifying the surface polarity of inorganic bodies is the sorption of a preformed polymer (typically having a rather high molecular weight, e.g., 3000 or more) of desired polarity/functionality onto a SiO2 support surface followed by crosslinking of the individual preformed polymer chains to one another to impart stability to the coating. Reversed-phase supports prepared in this fashion exhibit much improved low pH stability compared to those prepared by silylation. It is important to recognize, however, that the formation of a stable, crosslinked preformed polymer layer on the surface of the support does not reduce the need for a stable, inorganic support, since it is not possible to cover the entire inorganic surface. Although crosslinking of the polymer may keep it in place even as the underlying inorganic support dissolves, dissolution of the support will undoubtedly lead to a reduction in the mechanical stability of the support. In addition, problems related to increasing column back pressure are known to accompany the dissolution of the inorganic support and its subsequent appearance in the mobile phase and transport through the column and the accompanying instrumentation. Furthermore, the deposition of preformed polymers gives surfaces that are non-uniformly coated and can cause pore blockage of porous substrates.